Desiccation Tolerance of Adult Stem Cells in the Presence of Trehalose and Glycerol

Abstract

Development of protocols for storing desiccated cells at ambient temperatures offers tremendous economic and practical advantages over traditional storage procedures like cryopreservation and freeze-drying. As a first step for developing such procedures for adult stem cells, we have measured the post-rehydration membrane integrity (PRMI) of two passages, Passage-0 (P0) and Passage-1 (P1), of human adipose-derived stem cells (ASCs). ASCs were dried using a convective stage at three different drying rates (slow, moderate and rapid) in D-PBS with trehalose (50 mM) and glycerol (384 mM). ASCs were incubated in the drying media for 30 mins prior to drying at the prescribed rate on the convective stage for 30 mins. After drying, the ASCs were stored for 48 hrs in three different conditions: i) at ambient temperature, ii) in plastic bags at ambient temperature and iii) in vacuum sealed plastic bags at ambient temperature. PRMI was assessed after incubating the rehydrated ASCs with stromal medium for a further 48 hrs. Our measurements show that the PRMI of ASCs was: i) higher when ASCs were dried slowly; ii) increased when they were stored in vacuum as opposed to at ambient or in plastic bags; and iii) decreased with increasing passage of ASCs, i.e. under similar drying and storage conditions P0 ASCs had higher PRMI than P1 ASCs. Our results suggest that the best PRMI (37% for P0 ASCs and ~14% for P1 ASCs) can be achieved when the ASCs were dried slowly and stored in vacuum.

Enhancing long‐term storage and stability of engineered living materials through desiccant storage and trehalose treatment

“…Interestingly, addition of 10% glycerol resulted in a complete lack of post‐preservation viability (Figure 2e,f). While contradictory to prior accounts investigating cells (Mittal & Devireddy, 2008), glycerol's low melting temperature likely inhibited effective drying of the mixture (Horn et al, 2018). The persistence of a liquid‐like state during the freeze‐drying process is also suggested when comparing physical appearance of ELMs with and without glycerol (Figure 2c–f).…”

“…Beyond production, increased intracellular trehalose and proline accumulation can be achieved through exogenous addition via uptake by the general α‐glucoside transporter AGT1 (Han et al, 1995, p. 1) and proline‐specific permease/general amino acid permeases PUT4 and GAP1 (Lasko & Brandriss, 1981), respectively. The combination of lyoprotectants with exogenously added cryoprotectants such as glycerol can further improve cell viability following rehydration after cryopreservation (Storey et al, 1998) and desiccation (Mittal & Devireddy, 2008). Glycerol's low cost and ease of uptake by the STL1P importer in yeast cells makes it an attractive candidate for simple exogenous addition, with MAL62 overexpression also serving as a genetic alteration suitable for increasing intracellular glycerol accumulation (Sun et al, 2016).…”

Current Approaches of Preservation of Cells During (freeze-) Drying

“…Even if they did not particular address the question of the effect of glycerol, they could achieve results with 40% of cells still showing intact cell membranes. 62 After freeze-drying and reconstitution of bacteria, glycerol led to increased survival rates compared to water or maltodextrin, but still resulting in lower absolute numbers than trehalose or non-fat skim milk as drying excipients. 63 Bircher et al compared the effects of inulin, sucrose and glycerol on 6 different bacteria strains and reported enhanced viability (MPN method, optical density) after cryoconservation with glycerol, whereas enhanced viability after freeze-drying could be only observed for inulin and sucrose.…”

Recent Advances and Future Direction in Lyophilisation and Desiccation of Mesenchymal Stem Cells

“…Sorbitol was found to be enhanced protection on permeating through the plasma membrane, whose mechanism is yet to understand properly [ 26 ]. Glycerol also has a significant role in combination with trehalose in achieving higher postrehydration membrane integrity in the desiccation of adipose tissue derived adult stem cells [ 27 ].…”